The present invention relates to a system for the heterologous expression of a viral protein in a ciliate host cell.
As many viruses play a considerable role as human or animal pathogens, attention has been drawn to the use of viral proteins as vaccines against viral pathogens. Viruses (especially RNA viruses) are highly variable and many viral infections are due to viruses with multiple serotypes (e.g. Influenza virus, FMD virus). As a consequence, many of the existing viral vaccines are often unable to cope with the prevailing strains in the field and new ones have to be generated from the field strains with new outbreaks.
Despite the fact that, grace to modern biotechnology techniques, methods are available which allow for the heterologous expression of almost every conceivable protein, the expression of viral proteins is not such a common thing today, as viral proteins are, in most cases, structural proteins, i.e., neither enzymes nor protein hormones nor antibodies, and thus not that interesting for pharmaceutical or industrial purposes.
However, as many viruses play a considerable role as human or animal pathogens, attention has been drawn to the use of viral proteins as vaccines against viral pathogens.
Today, the standard procedure of producing vaccines against viral pathogens is to cultivate the respective virus in a given system, collect and inactivate virus particles thus obtained, and manufacture a pharmaceutical composition comprising said inactivated particles as a vaccine.
Said principle is for example practised in the production of influenza vaccine, which is being produced in fertilized chicken eggs. Eleven days after fertilization, the influenza virus strains are injected into the albumin of individual eggs and then infects the lungs of the developing embryo. After several days of incubation, the viruses are harvested and purified, chemically inactivated and used to produce a vaccine. On average, about one and two eggs are needed to produce one dose of vaccine. The entire production process lasts at least six months.
This way of vaccine production is well established and cost-effective, but has disadvantages as the procurement of many million eggs, the long timeline the tedious handling of the eggs and the limited flexibility in case of rapidly increasing demand or sudden appearance of new virus strains. Furthermore, fertilized chicken eggs can not be used to produce vaccines against a number of pathogenic virus, amongst them avian Influenza A virus (H5N1).
Anyway, fertilized chicken eggs can at least principally be used for the production of all viruses which are compatible with chicken embryoes as a host, or for the production of vaccines comprising elements of the former.
An alternative way of producing viruses, or vaccines comprising elements of the former is based on cell or tissue cultures. In one approach, the virus is injected into mammalian kidney cells. After propagation of the virus, the cells are lysed and then virus particles are harvested, purified and inactivated. However, these systems require the use of complex growing media, and their handling is laborious and time consuming. Furthermore, the said infection approach is difficult to carry out and not fully reproducible.
In another approach disclosed in WO03048348 a method for producing inactivated virus vaccines on the human cell line PER.C6® is disclosed. Because of the presence of the Sia2-6Gal and the Sia2-3Gal containing receptors on its surface this cell line is highly infectable with different viruses like Influenza virus, parainfluenza virus, adeno-associated virus or poliomavirus types. That might cause safety problems regarding the production process.
On the one hand mammalian cell culture based systems can, in contrast to the above egg based systems, be rapidly expanded and scaled up in times of emergency. On the other hand up-front costs for operational readiness of such production facilities (with its huge bioreactors) are much higher than the costs for egg-based systems, and the yield may be slightly slower. Again, these systems require the use of complex growing media, and their handling is laborious and time consuming.
Therefore, the development of alternative methods for the production of viral proteins which can be used as vaccines has high priority.
One approach is the production of those proteins using recombinant DNA techniques. One obvious advantage is a greatly improved safety of the vaccine, because of the opportunity to purify recombinant expressed viral protein in contrast to the vaccine production using eggs. Furthermore, the flexibility to adapt to different seasonal virus subtypes is highly increased.
However, as viral proteins are, in most cases structural proteins, the production for pharmaceutical and industrial purposes is complex and laborious. In the beginning it was attempted to express polypeptides corresponding to viral proteins like hemagglutinin in Escherichia coli. It is however noteworthy that heterologous expression of proteins which are to be used as vaccines in prokaryotes does not make sense, as the latter do not have a posttranslational modification apparatus. Therefore, proteins expressed in prokaryotes like Escherichia coli lack posttranslational modifications, like glycosylation pattern, which appears to affect the stability and contribute to a large extent to the immunogenic potential of an antigen. (Nayak et al. 1984).
Expression of viral proteins in the eukaryotic organism Saccharomyces cerevisiae (U.S. Pat. No. 4,752,473 for hemagglutinin) caused problems by the hyper-glycosylation of recombinant protein with the extensive addition of high molecular weight outer chain mannans (Jabbar and Nayak 1987, Jabbar et al. 1985) and had not proved satisfactory.
U.S. Pat. No. 5,858,368 discloses a method to express Influenza A virus hemagglutinin (HA) in recombinant baculovirus-infected insect cell lines. The proteins are chromatographically purified after being extracted from the peripheral membranes of the infected cells with a non-denaturing, nonionic detergent. The purification process is however rather time-consuming. Furthermore, it is questionable whether or not, due to their glycosylation pattern, proteins produced in a baculovirus/insect cell expression system are suitable for therapeutical use in mammals (Kulakosky et al. 1998). There are furthermore hints that certain residues in N-glycans of proteins expressed in insect cells represent potential allergenic epitopes (Tomiya et al. 2004).